Modified enzymes and their use for peptide synthesis

ABSTRACT

The present invention relates to modified enzymes with one or more amino acid residues from an enzyme being replaced by cysteine residues, where at least some of the cysteine residues are modified by replacing thiol hydrogen in the cysteine residue with a thiol side chain to form a modified enzyme, wherein the modified enzyme has high esterase and low amidase activity. Also, a method of producing the modified enzymes is provided. The present invention also relates to a method for using the modified enzymes in peptide synthesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No. 09/234,957, filed Jan. 21,1999, pending, and claims the benefit of U.S. Provisional PatentApplication No. 60/072,351, filed Jan. 23, 1998, abandoned, and U.S.Provisional Patent Application No. 60/072,265, filed Jan. 23, 1998.abandoned, the entire disclosures of which are hereby incorporated byreference in their entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to modified enzymes with one or more aminoacid residues being replaced by cysteine residues which are modified byreplacing thiol hydrogen in at least some of the cysteine residues witha thiol side chain to foam a modified enzyme. The modified enzyme hashigh esterase and low amidase activity. The present invention alsorelates to the use of modified enzymes in peptide synthesis.

BACKGROUND OF THE INVENTION

Modifying enzyme properties by site-directed mutagenesis has beenlimited to natural amino acid replacements, although molecularbiological strategies for overcoming this restriction have recently beenderived (Cornish et al., Angew. Chem., Int. Ed. Engl., 34:621-633(1995)). However, the latter procedures are difficult to apply in mostlaboratories. In contrast, controlled chemical modification of enzymesoffers broad potential for facile and flexible modification of enzymestructure, thereby opening up extensive possibilities for controlledtailoring of enzyme specificity.

Changing enzyme properties by chemical modification has been exploredpreviously, with the first report being in 1966 by the groups of Bender(Polgar et al., J. Am. Chem. Soc., 88:3153-3154 (1966)) and Koshland(Neet et al., Proc. Natl. Acad. Sci. USA, 56:1606-1611 (1966)), whocreated a thiolsubtilisin by chemical transformation (CH₂OH→CH₂SH) ofthe active site serine residue of subtilisin BPN′ to cysteine. Interestin chemically produced artificial enzymes, including some with syntheticpotential, was renewed by Wu (Wu et al., J. Am. Chem. Soc.,111:4514-4515 (1989); Bell et al., Biochemistry, 32:3754-3762 (1993))and Peterson (Peterson et al., Biochemistry. 34:6616-6620 (1995)), and,more recently, Suckling (Suckling et al., Bioorg. Med. Chem. Lett.,3:531-534 (1993)).

Enzymes are now widely accepted as useful catalysts in organicsynthesis. However, natural, wild-type, enzymes can never hope to acceptall structures of synthetic chemical interest, nor always be transformedstereospecifically into the desired enantiomerically pure materialsneeded for synthesis. This potential limitation on the syntheticapplicabilities of enzymes has been recognized, and some progress hasbeen made in altering their specificities in a controlled manner usingthe site-directed and random mutagenesis techniques of proteinengineering. However, modifying enzyme properties by protein engineeringis limited to making natural amino acid replacements and molecularbiological methods devised to overcome this restriction are not readilyamenable to routine application or large scale synthesis. The generationof new specificities or activities obtained by chemical modification ofenzymes has intrigued chemists for many years and continues to do so.

U.S. Pat. No. 5,208,158 to Bech et al. (“Bech”) describes chemicallymodified detergent enzymes where one or more methionines have beenmutated into cysteines. The cysteines are subsequently modified in orderto confer upon the enzyme improved stability towards oxidative agents.The claimed chemical modification is the replacement of the thiolhydrogen with C₁₋₆ alkyl.

Although Bech has described altering the oxidative stability of anenzyme through mutagenesis and chemical modification, it would also bedesirable to develop one or more enzymes with altered properties such asactivity, nucleophile specificity, substrate specificity,stereoselectivity, thermal stability, pH activity profile, and surfacebinding properties for use in, for example, detergents or organicsynthesis. In particular. enzymes, such as subtilisins, tailored forpeptide synthesis would be desirable. Enzymes useful for peptidesynthesis have high esterase and low amidase activities. Generally,subtilisins do not meet these requirements and the improvement of theesterase to amidase selectivities of subtilisins would be desirable.However, previous attempts to tailor enzymes for peptide synthesis bylowering amidase activity have generally resulted in dramatic decreasesin both esterase and amidase activities. Previous strategies forlowering the amidase activity include the use of water-miscible organicsolvents (Barbas et al., J. Am. Chem. Soc., 110:5162-5166 (1988); Wonget al., J. Am. Chem. Soc., 112:945-953 (1990); and Sears et al.,Biotechnol. Prog., 12:423-433 (1996)) and site-directed mutagenesis(Abrahamsen et al., Biochemistry, 30:4151-4159 (1991); Bonneau et al.,J. Am. Chem. Soc., 113:1026-1030 (1991); and Graycar et al., Ann. N.Y.Acad. Sci., 67:71-79 (1992)). However, while the ratios ofesterase-to-amidase activities were improved by these approaches, theabsolute esterase activities were lowered concomitantly. Abrahamsen etal., Biochemistry, 30:4151-4159 (1991). Chemical modification techniques(Neet et al., Proc. Nat. Acad. Sci., 56:1606 (1966); Polgar et al., J.Am. Chem. Soc., 88:3153-3154 (1966); Wu et al., J. Am. Chem. Soc.,111:4514-4515 (1989); and West et al., J. Am. Chem. Soc., 112:5313-5320(1990)), which permit the incorporation of unnatural amino acidmoieties, have also been applied to improve esterase to amidaseselectivity of subtilisins. For example, chemical conversion of thecatalytic triad serine (Ser221) of subtilisin to cysteine (Neet et al.,Proc. Nat. Acad. Sci., 56:1606 (1966); Polgar et al., J. Am. Chem. Soc.,88:3153-3154 (1966); and Nakatsuka et al., J. Am. Chem. Soc.,109:3808-3810 (1987)) or to selenocysteine (Wu et al., J. Am. Chem.Soc., 111:4514-4515 (1989)), and methylation of the catalytic triadhistidine (His57) of chymotrypsin (West et al., J. Am. Chem. Soc.,112:5313-5320 (1990)), effected substantial improvement inesterase-to-amidase selectivities. Unfortunately however, thesemodifications were again accompanied by 50-to 1000-fold decreases inabsolute esterase activity.

The present invention is directed to overcoming these deficiencies.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to modified enzymes with oneor more amino acid residues from an enzyme being replaced by cysteineresidues, where at least some of the cysteine residues are modified byreplacing thiol hydrogen in the cysteine residue with a thiol side chainto form a modified enzyme, where the modified enzyme has high esteraseand low amidase activity.

Another aspect of the present invention relates to a method of producinga modified enzyme. This method involves providing an enzyme with one ormore amino acids in the enzyme being replaced with cysteine residues andreplacing thiol hydrogen in at least some of the cysteine residues witha thiol side chain to form a modified enzyme. The modified enzyme hashigh esterase and low amidase activity.

The present invention also relates to a method of peptide synthesis.This method includes providing a modified enzyme with one or more aminoacid residues in the enzyme being replaced by cysteine residues, whereat least some of the cysteine residues are modified by replacing thiolhydrogen in the cysteine residue with a thiol side chain, where themodified enzyme exhibits high esterase and low amidase activity. An acyldonor, an acyl acceptor, and the modified enzyme are then combined underconditions effective to form a peptide product.

The modified enzymes of the present invention provide an alternative tosite-directed mutagenesis and chemical modification for introducingunnatural amino acids into proteins. In addition, these modified enzymesmore efficiently catalyze peptide synthesis as a result of an increasedesterase-to-amidase ratio compared to wild-type enzymes. Further, themodified enzymes of the present invention can incorporate D-amino acidesters as acyl donors in peptide synthesis and α-branched amides as acylacceptors in peptide synthesis to form a variety of dipeptides whichcannot be produced with wild-type (“WT”) enzymes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows peptide coupling catalyzed by an enzyme.

FIG. 2 shows the chemical modification of subtilisin Bacillus lentusmutants to generate chemically modified mutant enzymes.

FIG. 3 shows the ratio of k_(cat)/K_(M) constants foresterase-to-amidase activity. Esterase and amidase activity wasdetermined withsuccinyl-alanine-alanine-proline-phenylalanine-thiobenzyl ester(“suc-AAPF-SBn”) andsuccinyl-alanine-alanine-proline-phenylalanine-para-nitroanalide(“suc-AAPF-pNA”) substrates, respectively. All chemically modifiedmutants had the structure enzyme-CH₂—S—R, where the structure of thevarious R groups investigated is shown. In the N62C family, thestraight-chain alkyl group of intermediate length was hexyl (e) and inthe L217C family it was pentyl (d); n.d.=not determined. For comparison,the ratio for the WT enzyme was 17.

FIG. 4 shows the active site of subtilisin Bacillus lentus with sucAAPF(heavy black) bound. The catalytic triad and the four active siteresidues investigated are shown. Residue 62 is part of the S₂ pocket,residue 217 is at the mouth of the S₁′ (leaving group) pocket, residue166 is at the bottom of the S₁ pocket, and residue 222 is between the S₁and S₁′ pockets.

FIG. 5 shows the peptide ligation of L-amino acids using subtilisinBacillus lentus modified enzymes.

FIG. 6 shows the peptide ligation of D-amino acids using subtilisinBacillus lentus modified enzymes.

FIG. 7 shows the proposed binding of the Z-protecting group ofZ-D-Phe-Obn with subtilisin Bacillus lentus. The large hydrophobiccarbobenzoxy protecting (Z) group is binding in the S₁ pocket instead ofthe D-phenylalanine side chain.

FIG. 8 shows the chemical modification of S166C mutants of subtilisinBacillus lentus to generate modified enzymes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to modified enzymes with one or more aminoacid residues from an enzyme being replaced by cysteine residues, whereat least some of the cysteine residues are modified by replacing thiolhydrogen in the cysteine residue with a thiol side chain to form amodified enzyme. The modified enzyme has high esterase and low amidaseactivity.

Preferably, the enzyme is a protease. More preferably, the enzyme is aBacillus subtilisin. Subtilisins are alkaline serine proteases that arefinding increasing use in biocatalysis, particularly in chiralresolution, regioselective acylation of polyfunctional compounds,peptide coupling, and glycopeptide synthesis. The latter twoapplications are of particular interest, because they provide analternative to site-directed mutagenesis and chemical modification forintroducing unnatural amino acids into proteins. As shown in FIG. 1,subtilisins can catalyze peptide bond formation starting from an estersubstrate, by first forming an acyl enzyme intermediate which thenreacts with a primary amine to form the peptide product. Thisapplication thus requires high esterase activity to promote acyl enzymeformation and then low amidase activity to minimize hydrolysis of thepeptide bond of the desired product. Generally, subtilisins do not meetthese requirements and the improvement of the esterase to amidaseselectivities of subtilisins has been a long sought after goal.

Also, preferably, the amino acids replaced in the enzyme by cysteinesare selected from the group consisting of asparagine, leucine,methionine, or serine. More preferably, the amino acid to be replaced islocated in a subsite of the enzyme, preferably, the S₁, S₁′, or S₂subsites. Most preferably, the amino acids to be replaced are N62, L217,M222, and S166 where the numbered position corresponds tonaturally-occurring subtilisin from Bacillus amyloliquefaciens or toequivalent amino acid residues in other subtilisins, such as Bacilluslentus subtilisin.

In a particularly preferred embodiment, the enzyme is a Bacillus lentussubtilisin. In another particularly preferred embodiment, the amino acidto be replaced by cysteine is N62, L217, S166, or M222 and the thiolside chain group is selected from the group consisting of:

—SCH₃;

—SCH₂CH₃;

—SCH₂CH(CH₃)₂;

—S(CH₂)₄CH₃;

—S(CH₂)₅CH₃;

—S(CH₂)₉CH₃;

—SCH₂C₆H₅;

—SCH₂CH₂NH₃ ⁺; and

—SCH₂CH₂SO₃ ⁻; or

the amino acid to be replaced by cysteine is S166 or M222 and the thiolside chain group is selected from the group consisting of:

—SCH₂C₆H₅;

—SCH₂p-COOH—C₆H₄);

—SCH₂C₆F₅; and

—SCH₂CH₂NH₃ ⁺.

Preferably, the modified enzymes of the present invention have anesterase activity of from about 3.5 s⁻¹ mM⁻¹ to about 1110000 s⁻¹ mM⁻¹and an amidase activity of from about 0.056 s⁻¹ mM⁻¹ to about 35500 s⁻¹mM⁻¹. Most preferably, the modified enzymes of the present inventionhave an esterase activity from about 350 s⁻¹ mM⁻¹ to about 11100 s⁻¹mM⁻¹ and an amidase activity of from about 5.6 s⁻¹ mM⁻¹ to about 355 s⁻¹mM⁻¹.

A “modified enzyme” is an enzyme that has been changed by replacing anamino acid residue such as asparagine, serine, methionine, or leucinewith a cysteine residue and then replacing the thiol hydrogen of atleast some of the cysteine with a thiol side chain (e.g., —SCH₃,—SCH₂CH₃, —SCH₂CH(CH₃)₂, —S(CH₂)₄CH₃, —S) (CH₂)₅CH₃, —S(CH₂)₉CH₃,—SCH₂C₆H₅, —SCH₂CH₂NH₃ ⁺, —SCH₂CH₂SO₃ ⁻, —SCH₂(p-COOH—C₆H₄), and—SCH₂C₆F₅). After modification, the properties of the enzyme, i.e.,activity or substrate specificity, may be altered. Preferably, theactivity of the enzyme is increased.

The term “enzyme” includes proteins that are capable of catalyzingchemical changes in other substances without being changed themselves.The enzymes can be wild-type enzymes or variant enzymes. Enzymes withinthe scope of the present invention include pullulanases, proteases,cellulases, amylases, isomerases, lipases, oxidases, and reductases. Theenzyme can be a wild-type or mutant protease. Wild-type proteases can beisolated from, for example, Bacillus lentus or Bacillusamyloliquefaciens (also referred to as BPN′). Mutant proteases can bemade according to the teachings of, for example, PCT Publication Nos. WO95/10615 and WO 91/06637, which are hereby incorporated by reference.

Several types of moieties can be used to replace the thiol hydrogen ofthe cysteine residue. These include —SCH₃, —SCH₂CH₃, —SCH₂CH(CH₃)₂,—S(CH₂)₄CH₃, —S(CH₂)₅CH₃, —S(CH₂)₉CH₃; —SCH₂C₆H₅, —SCH₂CH₂NH₃ ⁺,—SCH₂CH₂SO₃ ⁻, —SCH₂(p-COOH—C₆H₄), and —SCH₂C₆F₅.

The terms “thiol side chain group,” “thiol containing group,” and “thiolside chain” are terms which are can be used interchangeably and includegroups that are used to replace the thiol hydrogen of a cysteine used toreplace one of the amino acids in an enzyme. Commonly, the thiol sidechain group includes a sulfur through which the thiol side chain groupsdefined above are attached to the thiol sulfur of the cysteine.

The binding site of an enzyme consists of a series of subsites acrossthe surface of the enzyme. The substrate residues that correspond to thesubsites are labeled P and the subsites are labeled S. By convention,the subsites are labeled S₁, S₂, S₃, S₄, S₁′, and S₂′. A discussion ofsubsites can be found in Siezen et al., Protein Engineering, 4:719-737(1991) and Fersht, Enzyme Structure and Mechanism, 2 ed., Freeman: NewYork, 29-30 (1985), which are hereby incorporated by reference. Thepreferred subsites are S₁, S₁′, and S₂.

Another aspect of the present invention relates to a method of producinga modified enzyme. This method involves providing an enzyme with one ormore amino acids in the enzyme being replaced with cysteine residues andreplacing thiol hydrogen in at least some of the cysteine residues witha thiol side chain to form a modified enzyme. The modified enzyme hashigh esterase and low amidase activity.

The amino acid residues of the present invention can be replaced withcysteine residues using site-directed mutagenesis methods or othermethods well known in the art. See, for example, PCT Publication No. WO95/10615, which is hereby incorporated by reference. One method ofmodifying the thiol hydrogen of the cysteine residue is set forth in theExamples.

The present invention also relates to a method of peptide synthesis.This method includes providing a modified enzyme with one or more aminoacid residues in the enzyme being replaced by cysteine residues, whereat least some of the cysteine residues are modified by replacing thiolhydrogen in the cysteine residue with a thiol side chain, where themodified enzyme exhibits high esterase and low amidase activity. An acyldonor, an acyl acceptor, and the modified enzyme are combined underconditions effective to form a peptide product.

Enzymatic peptide coupling is an attractive method for preparation of avariety of peptides, because this method requires minimal protection ofthe substrate, proceeds under mild conditions, and does not causeracemization. Wong et al., Enzymes in Synthetic Organic Chemistry,Pergamon Press: Oxford, 41-130 (1994), which is hereby incorporated byreference. In spite of these advantages, two major problems have limitedthe use of serine proteases in peptide synthesis. One is their efficientproteolytic (amidase) activity which causes hydrolysis of the couplingproduct, and the other is their stringent structural specificity andstereospecificity.

The modified enzymes of the present invention have alteredesterase-to-amidase activity as compared to the precursor enzyme.Increasing the esterase-to-amidase ratio enables the use of the enzymeto more efficiently catalyze peptide synthesis. In particular,subtilisins can catalyze peptide bond formation starting from an estersubstrate (i.e. an acyl donor), by first forming an acyl enzymeintermediate which then reacts with a primary amine (i.e. an acylacceptor) to form the peptide product, as shown in FIG. 1. This reactionthus requires high esterase activity to promote acyl enzyme formationand, then, low amidase activity to minimize hydrolysis of the peptidebond of the desired product. Modified enzymes of the present inventionshow an increased esterase-to-amidase ratio, without reducing theabsolute esterase activity of the enzyme. In addition, certain modifiedenzymes of the present invention even show a concomitant increase in theabsolute esterase activity.

Further. the modified enzymes of the present invention present asignificant enlargement of the applicability of chemically modifiedmutants of subtilisin Bacillus lentus in peptide synthesis. Thechemically modified mutant enzymes of the present invention canincorporate D-amino acid esters as acyl donors in peptide synthesis oran α-branched amino acid amide as acyl acceptor in peptide synthesis togive a variety of dipeptides. These reactions are not possible withsubtilisin Bacillus lentus-wild type (WT).

Therefore, the modified enzymes of the present invention can be used inorganic synthesis to, for example, catalyze a desired reaction and/orfavor a certain stereoselectivity. See e.g., Noritomi et al. Biotech.Bioeng. 51:95-99 (1996); Dabulis et al. Biotech. Bioeng. 41:566-571(1993), and Fitzpatrick et al. J. Am. Chem. Soc. 113:3166-3171 (1991),which are hereby incorporated by reference.

The modified enzymes of the present invention can be formulated intoknown powdered and liquid detergents having a pH between 6.5 and 12.0 atlevels of about 0.01 to about 5% (preferably 0.1% to 0.5%) by weight.These detergent cleaning compositions or additives can also includeother enzymes, such as known proteases, amylases, cellulases, lipases,or endoglycosidases, as well as builders and stabilizers.

The modified enzymes of the present invention, especially subtilisins,are useful in formulating various detergent compositions. A number ofknown compounds are suitable surfactants useful in compositionscomprising the modified enzymes of the present invention. These includenonionic, anionic, cationic, anionic, or zwitterionic detergents, asdisclosed in U.S. Pat. No. 4,404,128 to Anderson and U.S. Pat. No.4,261,868 to Flora et al., which are hereby incorporated by reference. Asuitable detergent formulation is that described in Example 7 of U.S.Pat. No. 5,204,015 to Caldwell et al., which is hereby incorporated byreference. The art is familiar with the different formulations which canbe used as cleaning compositions. In addition to typical cleaningcompositions, it is readily understood that the modified enzymes of thepresent invention may be used for any purpose that native or wild-typeenzymes are used. Thus, these modified enzymes can be used, for example,in bar or liquid soap applications, dishcare formulations, contact lenscleaning solutions or products, peptide synthesis, feed applicationssuch as feed additives or preparation of feed additives, wastetreatment, textile applications such as the treatment of fabrics, and asfusion-cleavage enzymes in protein production. The modified enzymes ofthe present invention may achieve improved wash performance in adetergent composition (as compared to the precursor). As used herein,improved wash performance in a detergent is defined as increasingcleaning of certain enzyme-sensitive stains such as grass or blood, asdetermined by light reflectance evaluation after a standard wash cycle.

The addition of the modified enzymes of the present invention toconventional cleaning compositions does not create any special uselimitation. In other words, any temperature and pH suitable for thedetergent is also suitable for the present compositions as long as thepH is within the above range and the temperature is below the describedmodified enzyme's denaturing temperature. In addition, modified enzymesin accordance with the invention can be used in a cleaning compositionwithout detergents, again either alone or in combination with buildersand stabilizers.

In another aspect of the present invention, the modified enzymes areused in the preparation of an animal feed, for example, a cereal-basedfeed. The cereal can be at least one of wheat, barley, maize, sorghum,rye, oats, triticale, and rice. Although the cereal component of acereal-based feed constitutes a source of protein, it is usuallynecessary to include sources of supplementary protein in the feed suchas those derived from fish-meal, meat-meat, or vegetables. Sources ofvegetable proteins include at least one of full fat soybeans, rapeseeds,canola, soybean-meal, rapeseed-meal, and canola-meal.

The inclusion of a modified enzyme of the present invention in an animalfeed can enable the crude protein value and/or digestibility and/oramino acid content and/or digestibility coefficients of the feed to beincreased, which permits a reduction in the amounts of alternativeprotein sources and/or amino acids supplements which had previously beennecessary ingredients of animal feeds.

The feed provided by the present invention may also include other enzymesupplements such as one or more of β-glucanase, glucoamylase, mannanase,α-galactosidase, phytase, lipase, α-arabinofuranosidase, xylanase,α-amylase, esterase, oxidase, oxido-reductase, and pectinase. It isparticularly preferred to include a xylanase as a further enzymesupplement such as a subtilisin derived from the genus Bacillus. Suchxylanases are, for example, described in detail in PCT PatentApplication No. WO 97/20920, which is hereby incorporated by reference.

Another aspect of the present invention is a method for treating atextile. The method includes providing a modified enzyme with one ormore amino acid residues from an enzyme being replaced by cysteineresidues, wherein the cysteine residues are modified by replacing thiolhydrogen in at least some of the cysteine residues with a thiol sidechain to form a modified enzyme, where the modified enzyme has highesterase and low amidase activity. The modified enzyme is contacted witha textile under conditions effective to produce a textile resistance tocertain enzyme-sensitive stains. Such enzyme-sensitive stains includegrass and blood. Preferably, the textile includes a mutant enzyme. Themethod can be used to treat, for example, silk or wool as described inpublications such as Research Disclosure 216,034, European PatentApplication No. 134,267, U.S. Pat. No. 4,533,359, and European PatentApplication No. 344,259, which are hereby incorporated by reference.

EXAMPLES Example 1 Producing the Cys-Mutants

The gene for subtilisin from Bacillus lentus (“SBL”) was cloned into thebacteriophage M13mp19 vector for mutagenesis (U.S. Pat. No. 5,185,258,which is hereby incorporated by reference). Oligonucleotide-directedmutagenesis was performed as described in Zoller et al., MethodsEnzymol., 100:468-500 (1983), which is hereby incorporated by reference.The mutated sequence was cloned, excised, and reintroduced into theexpression plasmid GG274 in the B. subtilis host. PEG (50%) was added asa stabilizer. The crude protein concentrate obtained was purified byfirst passing through a Sephadex™ G-25 desalting matrix with a pH 5.2buffer (20 mM sodium acetate, 5 mM CaCl₂) to remove small molecularweight contaminants. Pooled fractions for the desalting column were thenapplied to a strong cation exchange column (SP Sepharose™ FF) in thesodium acetate buffer (above), and SBL was eluted with a one stepgradient of 0-200 mM NaCl acetate buffer, pH 5.2. Salt-free enzymepowder was obtained following dialysis of the eluent against Milliporepurified water, and subsequent lyophilization. The purity of the mutantand wild-type enzymes, which had been denatured by incubation with 0.1 MHCl at 0° C. for 30 minutes, was ascertained by SDS-PAGE on homogeneousgels using the Phast™ System from Pharmacia (Uppsala, Sweden). Theconcentration of SBEL was determined using the Bio-Rad (Hercules,Calif.) dye reagent kit which is based on the method of Bradford,Analytical Biochemistry, 72:248-254 (1976), which is hereby incorporatedby reference. Specific activity of the enzymes was determined in pH 8.6buffer using the method described in Example 3 below.

Example 2 Preparation of Certain Moieties Preparation of 2, 3, 4, 5,6-pentafluorobenzyl methanethiosulfonate

2, 3, 4, 5, 6-pentafluorobenzyl methanethiosulfonate was preparedaccording to the general procedure in Examples 1 and 3 fromα-bromo-2,3,4,5,6-pentafluorotoluene in 88% yield. m.p.: 64.2-64.7° C.(95% EtOH);IR (KBr): 3030, 3009, 2961, 2930, 2920, 1514, 1314, 1132,980, 880, and 748 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ4.46 (br s, 2H, SCH₂),3.36 (s, 3H, CH₃SO₂); MS (El): 292 (M⁺), 212 (⁺S═CHC₆F₅); HRMS (El)291.9648 (M⁺, calc'd for C₈H₅F₅O₂S₂: 291.9651).

Preparation of 4-carboxybenzyl methanethiosulfonate

4-carboxybenzyl methanethiosulfonate was prepared according to thegeneral procedure in Examples 1 and 3 from α-bromo-p-toluic acid in 60%yield after recrystallization from 95% EtOH. m.p.:187.6-187.8° C.; IR(KBr): 3300-2200, 1683, 1608, 1577, 1422, 1301, 1180, 1121, 957, 863,750, 716, and 551 cm⁻¹; ¹H NMR (200 MHz, DMSO-d₆ and 10% D₂O): δ7.90 (d,J=8.0 Hz, 2H, aromatic), 7.51 (d, J=8.0 Hz, 2H, aromatic), 4.47 (s, 2H,SCH₂), 3.23 (3H, CH₃SO₂S); ¹³C NMR (50 MHz, DMSO-d₆ and 10% D₂O):δ167.87, 141.55, 130.56, 130.45, 130.07, 51.01, 39.37; MS (El): 246(M⁺), 229 (M⁺)—OH), 166 (base peak, ⁺S═CH—Ar); HRMS (El): 246.0031 (M⁺,calc'd. for C₉H₁₀O₄S₂: 246.0021).

Example 3 Peptide Synthesis Using Modified Enzymes with Alkyl MoietiesMaterials

Succinyl-alanine-alanine-proline-phenylalanine-para-nitroanalide(“suc-AAPF-pNA”) andsuccinyl-alanine-alanine-proline-phenylalanine-thiobenzyl ester(“suc-AAPF-SBn”) were both from Bachem Inc. (Torrance, Calif.), Ellman'sreagent (5,5′-dithiobis-2,2′-nitrobenzoic acid, DTNB) andphenylmethanesulfonyl fluoride (“PMSF”) were from Sigma-Aldrich Inc.(Milwaukee, Wis.). Sources and syntheses of methanethiosulfonate (“MTS”)reagents were as described in Berglund et al., J. Am. Chem. Soc.,119:5265-5266 (1997), which is hereby incorporated by reference.Buffers, 2-[N-cyclohexylamino]ethanesulfonic acid (CHES), 4-morpholineethanesulfonic acid (MES) and tris hydroxymethylaminomethane(Tris) were from Sigma-Aldrich Inc. (Milwaukee, Wis.). Wild type SBL andcysteine mutants N62C, S166C, L217C, and M222C were provided by GenencorInternational Inc., Rochester, N.Y. and purified as described in Example1 and Stabile et al., Bioorg. Med. Chem. Lett., 6:2501-2506 (1996),which is hereby incorporated by reference.

Chemical Modification

Chemical modification with alkyl MTS reagents was carried out asdescribed in Berglund et al., J. Am. Chem. Soc., 119:5265-5266 (1997)and DeSantis et al., Biochemistry, 37:5968-5973 (1998), which are herebyincorporated by reference. Briefly, 200 μL of a 1 M solution of MTSreagent in a suitable solvent was added to a solution (5-10 mg/mL, 3.5mL) of the cysteine mutant in 70 mM CHES, 5 mM MES, 2 mM CaCl₂ pH 9.5.The MTS reagent was added in two portions over 30 minutes. Reactionmixtures were kept at 20° C. with continuous end-over-end mixing.Reactions were monitored by following the specific activity withsuc-AAPF-pNA and by tests for residual free thiol with Ellman's reagent.Once the reaction was complete, the reaction mixture was loaded on aSephadex PD-10 G25 column with 5 mM MES and 2 mM CaCl₂, pH 6.5. Theprotein fraction was dialyzed against 1 mM CaCl₂, and the dialysate waslyophilized.

Characterization of Modified Enzymes

The molecular mass of each modified enzyme (“ME”) was determined byelectrospray ionization mass spectrometry (Berglund et al., J. Am. Chem.Soc., 119:5265-5266 (1997); DeSantis et al., Biochemistry, 37:5963-5973(1998), which are hereby incorporated by reference). The purity of theMEs was ascertained by native PAGE on 8-25% gels using the Phast systemfrom Pharmacia (Uppsala, Sweden). The extent of chemical modification ofthe cysteine mutants was determined by thiol titration with DTNB for the62, 217 and 166 mutants and with I₂ (Cunningham et al., J. Biol. Chem.,234:1447-1451 (1959), which is hereby incorporated by reference) for themore sterically hindered 222 mutants which do not react with DENS.Active site titrations were performed on all enzymes by monitoring theburst of fluoride released upon addition of phenylmethanesulfonylfluoride to the enzyme, as described in Hsia et al., J. Anal. Biochem.,242:221-227 (1996), which is hereby incorporated by reference.

Rapid Screen on Microtiter Plates

Detailed procedures and validation of this assay have been described inPlettner et al., Bioorg. Med. Chem. Lett., 8:2291-2296 (1998), which ishereby incorporated by reference. Briefly, enzyme solutions wereprepared in 5 mM MES with 2 mM CaCl₂, pH 6.5 at about 10⁻⁷ M for amidaseand 10⁻⁸ M for esterase. Substrate solutions in DMSO were 1.6 mM(amidase) and 1.0 mM (esterase). The assay was performed at pH 8.6 inthe same buffer used for kinetics (see below). Enzyme solutions werearranged on a microtiter plate (loading plate) along columns, with thelast well in each column as a buffer blank. On a separate plate (assayplate), 10 μL of substrate and 180 μL of buffer was added to each well.Reactions were initiated by transferring 10 μL of enzyme from anappropriate column on the loading plate to the assay plate. Reactionswere monitored on a Multiscan MCC 340 96-well reader programmed in thekinetic mode at 414 nm, with no time lag, at 5 second intervals for atotal time of 1 minute (amidase) and 30 seconds (esterase). Backgroundhydrolysis was subtracted automatically. The k_(cat)/K_(M) was estimatedfrom the rate of substrate hydrolysis (v) using the low-substrateapproximation: v≈k_(cat)/K_(M) [E][S] where, [S]<<K_(M).

Kinetics

Assays were done in 0.1 M Tris pH 8.6 containing 0.005% Tween. Substratesolutions were prepared in DMSO. In the esterase assay, substratesolutions also contained 0.0375 M DTNB (Bonneau et al., J. Am. Chem.Soc., 113:1026-1030 (1991), which is hereby incorporated by reference).Concentrations of substrate stock solutions ranged from 0.013 to 0.3 Mfor amidase and 0.0015 to 0.3 M for esterase, and 9-10 differentconcentrations were tested in duplicate for each enzyme. Enzymesolutions were prepared in 20 mM MES, 1 mM CaCl₂, pH 5.8, at aconcentration of 10⁻⁶ M for amidase and 10⁻⁷ M for esterase. Reactionswere monitored spectrophotometrically on a Perkin Elmer Lambda 2instrument equipped with a thermostatted cell compartment.

Prior to an assay, 980 μL of Tris buffer in a cuvette was equilibratedto 25° C. Substrate stock solution (10 μL) was added to the buffer andthe reading set to zero. Reactions were initiated by addition of 10 μLof enzyme solution and were monitored at 410 nm (amidase) and 412 nm(esterase). Extinction coefficients for the chromophores were 8800 M⁻¹cm⁻¹ for p-nitroaniline (Bonneau et al., J. Am. Chem. Soc.,113:1026-1030 (1991), which is hereby incorporated by reference) and13470 M⁻¹ cm⁻¹ for 3-carboxylate-4-nitrothiophenolate in 0.1 M Tris pH8.6 with 0.005% Tween. Initial rates were obtained by linear fitting upto 5% conversion; r values exceeded 0.9996. In the case of esterase,rates in the presence of enzyme were corrected for uncatalyzedbackground hydrolysis of the thiobenzyl ester. Kinetic constants wereobtained by fitting the rate data to the Michaelis-Menten equation usingGrafit.® (Erithacus Software Ltd., Staines, Middlesex, United Kingdom)

Reaction of the Cysteine Mutants with DTNB

Since [DTNB]>>[enzyme] and [DTNB]≈constant over 30 seconds (time for 5%conversion), the pseudo-first order rate constant for the reaction ofN62C, L217C and S166C mutants with DTNB was determined under the sameconditions as used in the assay, using enzyme concentrations from 10⁻⁶to 10⁻⁴ M. The pseudo first-order rates constant of reaction of N62C,L217C, and S166C with DTNB under the esterase assay conditions were1.8×10⁻⁴ s⁻¹ (0.5%=maximum amount of cysteine mutant reacted with DTNBover the time of the esterase assay), 1.4×10⁻³ s⁻¹ (4.2% reacted), and1.4×10⁻⁴ S⁻¹ (0.4% reacted), respectively. The M222C mutant did notdetectably react with DTNB.

Results

Each of the N62C, L217C, S166C, and M222C mutants of SBL were preparedand purified, and the introduced —CH₂SH side-chain specifically andquantitatively chemically modified with the MTS reagents with alkylmoieties —SCH₃, —SCH₂CH₃, —SCH₂CH(CH₃)₂, —S(CH₂)₄CH₃, —S(CH₂)₅CH₃,—S(CH₂)₉CH₃, —SCH₂C₆H₅, —SCH₂CH₂NH₃ ⁺, —SCH₂CH₂SO₃ ⁻ (-a-i), asdescribed previously (Berglund et al., Bioorg Med. Chem. Lett.,6:2507-2512 (1996); Berglund et al., J. Am. Chem. Soc., 119:5265-5266(1997); DeSantis et al., Biochemistry, 37:5968-5973 (1998); and DeSantiset al., J. Am. Chem. Soc., 120:8582-8586 (1998), which are herebyincorporated by reference). The purities of the MEs generated wereestablished by native polyacrylamide gel electrophoresis (PAGE), whichshowed only one band in each case, thereby demonstrating that the MEswere pure and that dimerization had not occurred. Mass analyses of theMEs by electrospray mass spectrometry were consistent (±6 Da) with thecalculated masses for single-site modifications. Berglund et al., J. Am.Chem. Soc., 119:5265-5266 (1997) and DeSantis et al., Biochemistry,37:5968-5973 (1998). Titration of the N62C, S166C, and L217C MEs withEllman's reagent showed a residual thiol content of less than 2% in allcases, confirming that the MTS reactions were virtually quantitative.Ellman et al., Biochem. Pharmacol., 7:88-95 (1961), which is herebyincorporated by reference. The residual free thiol content for the moresterically hindered M222C MEs, which did not react with Ellman'sreagent, was determined with I₂ (Cunningham et al., J. Biol. Chem.,234:1447-1451 (1959)). The M222C MEs contained ≦2% free thiol, exceptfor M222C—SCH₂CH₂SO₃ ⁻ (-i) which contained 3% residual thiol groups.The concentration of active enzyme was determined by active sitetitration with phenylmethanesulfonyl fluoride (PMSF). Hsia et al., J.Anal. Biochem., 242:221-227 (1996). All of the MEs were 60-80% active byweight, except for M222C—SCH₂CH₂SO₃ ⁻ (-i) which contained only 4%active enzyme and was. therefore, not investigated further.

Initially, a rapid screen on microtiter plates (Plettner et al., Bioorg.Med. Chem. Lett., 8:2291-2296 (1998), which is hereby incorporated byreference) was used to generate estimates of k_(cat)/K_(M) for amidaseand esterase for the enzymes outlined in FIG. 2. Of 36 MEs and fourcysteine mutants screened, 25 enzymes were chosen for further kineticanalyses. These included all the promising esterases, as well as a fewmutants with severely damaged esterase activity for comparison. Theresults of the kinetic analyses with suc-AAPF-pNA and suc-AAPF-SBn asstandard amide and ester substrates respectively, are presented in Table1, below. It was recognized that the cysteine thiol of the unmodifiedcysteine mutants N62C, L217C, S166C, and M222C could react with DTNB,which is used in the kinetic assay to detect the thiol benzyl hydrolysisproduct of the esterase reaction. This possibility was discounted bystudying the rates of reaction of DTNB with N62C, S166C, L217C, andβ-mercaptoethanol as a model for a non-hindered thiol which establishedthat these did not react at a rate sufficient to interfere with theassay at the concentrations used.

TABLE 1 Kinetic constants of chemically modified mutants for amidase andesterase activities Amidase^(a) Esterase^(b) k_(cat)/K_(M) k_(cat)/K_(M)Enzyme k_(cat) (s⁻¹)^(c) K_(M) (mM)^(c) (s⁻¹ mM⁻¹) k_(cat) (s⁻¹)^(c)K_(M) (mM)^(c) (s⁻¹ mM⁻¹) WT 153 ± 4  0.73 ± 0.05 209 ± 15 1940 ± 1800.54 ± 0.07  3560 ± 540^(d) N62C 163 ± 8  1.9 ± 0.2  86 ± 10 2370 ± 90 0.54 ± 0.06 4380 ± 510 N62C-S-a 73 ± 2 0.55 ± 0.04 133 ± 10 3130 ± 90 0.31 ± 0.03 10100 ± 1000 N62C-S-b 97 ± 2 0.55 ± 0.04 177 ± 13 2220 ± 110 0.2 ± 0.04 11100 ± 2300 N62C-S-c 139 ± 4  0.75 ± 0.06 185 ± 16 2180 ±80  0.25 ± 0.04  8700 ± 1430 N62C-S-e 146 ± 7  0.63 ± 0.08 230 ± 30 2330± 150 0.26 ± 0.06  8970 ± 2150 N62C-S-f 124 ± 4  0.36 ± 0.04 344 ± 401000 ± 47  0.39 ± 0.06 2570 ± 410 N62C-S-g 121 ± 3  0.34 ± 0.03 355 ± 331840 ± 110 0.29 ± 0.06  6330 ± 1360 N62C-S-h 96 ± 5 1.0 ± 0.1  98 ± 112660 ± 80  0.48 ± 0.04 5540 ± 490 N62C-S-i 111 ± 4  0.93 ± 0.07 120 ± 103190 ± 110 0.61 ± 0.06 5230 ± 540 L217C 38 ± 1 0.80 ± 0.04 48 ± 3 3160 ±120 0.57 ± 0.06 5540 ± 620 L217C-S-a 47 ± 2 0.62 ± 0.07 76 ± 9 2520 ±120 0.56 ± 0.07 4500 ± 600 L217C-S-c 93 ± 2 0.61 ± 0.03 152 ± 8  2450 ±70  0.31 ± 0.03 7900 ± 800 L217C-S-d 87 ± 3 0.52 ± 0.05 167 ± 17 2280 ±80  0.39 ± 0.04 5840 ± 640 L217C-S-f 120 ± 3  0.54 ± 0.03 223 ± 13 1840± 100 0.50 ± 0.08 3690 ± 620 L217C-S-h 36 ± 1 0.64 ± 0.06 56 ± 6 3070 ±90  0.41 ± 0.04 7490 ± 760 L217C-S-i 83 ± 6 1.8 ± 0.2 47 ± 6 5060 ± 1301.0 ± 0.1 5060 ± 520 S166C 42 ± 1 0.50 ± 0.05 84 ± 9 600 ± 70 1.7 ± 0.4350 ± 90 S166C-S-a 46 ± 2 0.34 ± 0.05 135 ± 20 2320 ± 50  0.38 ± 0.036100 ± 500 S166C-S-g   23 ± 0.5 1.2 ± 0.1 20 ± 1 1530 ± 110 0.31 ± 0.08 4900 ± 1300 S166C-S-h 50 ± 1 0.68 ± 0.04 74 ± 5 1350 ± 50  0.61 ± 0.072200 ± 270 S166C-S-i 25 ± 1 1.3 ± 0.1 19 ± 1 1950 ± 90  1.9 ± 0.2 1030 ±120 M222C 61 ± 2 0.81 ± 0.07 75 ± 6 3080 ± 140 0.58 ± 0.07 5300 ± 680M222C-S-a 56 ± 2 0.91 ± 0.07 62 ± 6 2090 ± 120 1.3 ± 0.2 1610 ± 270M222C-S-h  5.0 ± 0.2 0.91 ± 0.08  5.6 ± 0.9 1970 ± 140 0.4 ± 0.1  4920 ±1280 ^(a)substrate: suc-AAPF-pNA; ^(b)substrate; sucAAPF-SBn;^(c)determined by the method of initial rates; ^(d)mean standard three(esterase) experiments.

The broad applicability of the chemical modification approach forachieving the goal of improved esterase-to-amidase selectivity withoutreducing absolute esterase activity is evident from the Table 1 datasince of 25 MEs and cysteine mutants evaluated, fully 19 displayedimproved esterase to amidase selectivity. Furthermore, 20 displayedesterase activity that was higher than WT (See FIG. 3).

Of the N62 MEs, all except N62C—S(CH₂)₉CH₃ (-f) exhibited improvedesterase activity relative to WT. Even the N62 mutation to cysteineitself created a better esterase and poorer amidase than WT. Chemicalmodification of N62C enhanced the absolute esterase activity stillfurther, to ≈3-fold greater than WT for N62C—S—CH₃, (-a) andN62C—SCH₂CH₃, (-b). In fact, N62C—SCH₂CH₃, (-b) with its k_(cat)/K_(M)of 11100±2300 s^(−b 1) mM⁻¹ had the highest absolute esterase activityof all the MEs investigated. However, the larger R groups ofN62C—SCH₂CH(CH₃)₂ (-c) to N62C—SCH₂C₆H₅ (-g) caused decreases in k_(cat)and k_(cat)/K_(M) for esterase catalysis, and steady increases in bothk_(cat) and k_(cat)/K_(M) for amidase. Consequently, the ratio ofk_(cat)/K_(M) for esterase to amidase activity decreased 10-fold as thechain length of -R increased from N62C—S—CH₃ (-a) to N62C—S(CH₂)₉CH₃(-f) (FIG. 3). The positively and negatively charged MEs,N62C—SCH₂CH₂NH₃ ⁺ (-h) and N62C—SCH₂CH₂SO₃ ⁻ (-i) respectively, bothexhibited higher esterase and lower amidase activity than WT, with theimprovement in the esterase-to-amidase ratio being ≈3-fold regardless ofthe sign of the charge introduced. In addition, the larger R groups ofN62C—SCH₂CH(CH₃)₂ to N62C—SCH₂C₆H₅ (-c to -g) elicited reduced K_(M)sfor both ester and amide substrates. This demonstrates that hydrophobicinteractions at the 62 site are beneficial to binding.

All of the L217C CMMs generated also exhibited improved esterasek_(cat)/K_(M)s compared to WT. At this site, mutation to cysteine aloneagain generated a superior catalyst having 1.5-fold better esterase and4-fold poorer amidase activity than WT. However, its modification toL217C—S—CH₃ (-a) caused a decrease in both esterase k_(cat) andk_(cat)/K_(M) compared to L217C itself. L217C—SCH₂CH(CH₃)₂ (-c) was themost active 217 esterase and exhibited a k_(cat)/K_(M) of 7900±800 s⁻¹mM⁻¹. While all of the 217 MEs exhibited greater than WT esteraseactivity, further increases in the chain length of -R from —S(CH₂)₄CH₃to —S(CH₂)₉CH₃, (-d to -f caused further decreases in k_(cat) andk_(cat)/K_(M). This is in contrast to the trend observed for amidasek_(cat) and k_(cat)/K_(M), values for the same MEs. Berglund et al., J.Am. Chem. Soc., 119:5265-5266 (1997), which is hereby incorporated byreference. As a result, all of the L217C MEs except L217C—S(CH₂)₉CH₃(-f) had higher than WT esterase to amidase selectivity (FIG. 3). Thepositively charged L217C—SCH₂CH₂NH₃ ⁺ (-h) and negatively chargedL217C—SCH₂CH₂SO₃ ⁻ (-i) MEs also displayed higher than WT esteraseactivities, with L217C—SCH₂CH₂SO₃ ⁻ (-i) having a 2.6-fold higher thanWT esterase k_(cat). Furthermore, at 5060 s⁻¹, this was the highestesterase k_(cat) of all the MEs studied. The L217C—SCH₂CH₂NH₃ ⁺ (-h) hada (k_(cat)/K_(M)) _(ester)/(k_(cat)/K_(M))_(amide) ratio of 134,compared to 17 for WT. The correlations between decreased esterase andincreased amidase activities with increasing chain length of -R, andimproved esterase and decreased amidase for charged modifications,paralleled each other for both the L217C and N62C MEs. These equivalenttrends are consistent with residues 217 and 62 being equidistant fromHis 64 of the catalytic triad (See FIG. 4).

Modification of the S166C residue of the S₁ pocket, which is quiteremote from the catalytic triad and from the S₁′ leaving group site ofboth the ester or amide substrates, exerted large effects onesterase-to-amidase selectivity. The S166C mutant itself, with ak_(cat)/K_(M) of 350 s⁻¹ mM⁻¹, had the lowest esterase activity of allthe MEs evaluated. However, it also had somewhat decreased amidaseactivity, giving an esterase-to-amidase selectivity ratio of four,compared to 17 for WT. Apart from having the lowest k_(cat) foresterase, S166C had a significantly higher K_(M) for esterase than theWT and was one of few mutants for which K_(M) (esterase)>K_(M)(amidase). In contrast, modification of S166C to generate S166C—S—CH₃(-a) increased esterase-to-amidase selectivity to 45, a ≈3-foldimprovement relative to WT. The large hydrophobic benzyl group ofS166C—S—CH₂C₆H₅ (-g) increased esterase-to-amidase selectivity stillfurther to 245, which was 14-fold higher than WT, while the chargedhydrophilic groups of S166C—SCH₂CH₂NH₃ ⁺ (-h) and S166C—SCH₂CH₂SO₃ ⁻ (i)induced little improvement in the esterase-to-amidase ratio. That theesterase K_(M) decreased, while the amidase K_(M) increasedsignificantly, relative to WT for the S166C—S—CH₂C₆H₅ (-g) ME, impliedlong-range interactions between its S₁ and S₁′ pockets and differentrate-determining steps. These results complement those previouslyobserved for the more hydrophilic G166N and G166S mutants of subtilisinBPN′, both of which effected improved esterase and esterase-to-amidaseactivity relative to WT. Bonneau et al., J. Am. Chem. Soc., 1131026-1030 (1991), which is hereby incorporated by reference.

At the Met222 site, both M222C—SCH₂CH₂NH₃ ⁺ (-h) and M222C exhibited animproved esterase k_(cat)/K_(M) of up to 1.5, while all of M222C—S—CH₃(-a), M222C—SCH₂CH₂NH₃ ⁺ (-h), and M222C displayed up to 37-fold reducedamidase activity. The esterase-to-amidase activity of the cysteineparent, M222C, with its 4-fold improvement, was itself significantlyhigher than WT. The M222C mutant has a S₁′ leaving group site that isless sterically congested than WT. This may enhance the rate ofacyl-enzyme hydrolysis, which is often the rate-determining step forester substrates. M222C—S—CH₃ (-a), which differs from WT only in thereplacement of one of the methionine side-chain methylenes (CH₂) bysulfur, had the same k_(cat) as WT, but an increased K_(M). At thissite, the most improved ME was M222C—SCH₂CH₂NH₃ ⁺ (-h), which exhibitedan esterase-to-amidase selectivity of 879, compared to 17 for the WT.This 52-fold improvement in esterase-to-amidase ratio of the seriesarose largely from a 31-fold lowered amidase k_(cat), but with the WTlevel of esterase k_(cat) being retained. This result was consistentwith the observation that the M222K mutant of subtilisin BPN′ causedimproved esterase activity and severely decreased amidase activity,thus, generating an enzyme with greatly improved esterase-to-amidasespecificity. Graycar et al., Ann. N.Y. Acad. Sci., 672:71-79 (1992),which is hereby incorporated by reference.

With 19 of 25 MEs evaluated achieving the goal of better-than-WTesterase-to-amidase selectivity without diminishing the absoluteesterase-rate, the ME approach was clearly broadly applicable. Overall,esterase-to-amidase specificity varied from 4-fold lower than WT forS166C to 52-fold higher than WT for M222C—SCH₂CH₂NH₃ ⁺. At least onemember from each of the four families of mutants studied met bothcriteria of excellent esterase activity and high esterase-to-amidaseselectivity, with: N62C—SCH₃, (-a) being 3-fold, L217C—SCH₂CH₂NH₃ ⁺ (-h)and L217C—SCH₂CH₂SO₃ ⁻ (-i) 6 to 8-fold, S166C—SCH₂C₆H₅ (-g) was14-fold, and M222C—SCH₂CH₂NH₃ ⁺ (-h) was 52-fold improved in terms ofesterase-to-amidase ratio relative to the WT enzyme. With up to 880-foldesterase to amidase selectivity achievable by the ME approach, thepotential of chemically modified mutant subtilisins for peptidesynthesis was expanded considerably.

Example 4 Peptide Synthesis using S166C—SCH₂C₆H₅,S166C—SCH₂(p-COOH—C₆H₄), S166C—SCH₂C₆F₅, S166C—SCH₂CH₂NH₃ ⁺, andM222C—SCH₂CH₂NH₃ ⁺. General Methods

WT-subtilisin Bacillus lentus and mutant enzymes, S166C and M222C werepurified (Stabile et al., Bioorg. Med. Chem. Lett., 6:2501-2506 (1996);Berglund et al., Bioorg. Med. Chem. Lett., 6:2507-2512 (1996); andDeSantis et al., Biochemistry, 37:5698-5973 (1998), which are herebyincorporated by reference) and prepared as previously reported inDeSantis et al., Biochemistry, 37:5698-5973 (1998), which is herebyincorporated by reference. Protected amino acids were purchased fromSigma or Bachem and were used as received. All solvents were reagentgrade and distilled prior to use. Thin layer chromatography analysis andpurification were performed on pre-coated Merck Silica gel (60 F-254)plates (250 μm) visualized with UV light or iodine. ¹H and ¹³C NMRspectra were recorded on a Varian Gemini 200 (200 MHz for ¹H and 50.3MHz for ¹³C) or Unity 400 (400 MHz for ¹H and 100 MHz for ¹³C) andspectrometer and chemical shifts are given in ppm (δ) using CDCl₃ orDMSO-d₆ as an internal standard. High resolution mass spectra (HRMS)were recorded using Micromass ZAB-SE (FAB⁺) Optical rotations weremeasured with a Perkin-Elmer 243B polarimeter.

General Procedure for Peptide Ligation

To a solution of amino acid acyl donor (0.1 mmol) in DMF (0.4 mL) andwater (0.4 mL), glycinamide hydrochloride (0.3 ) mmol) or alaninamidehydrochloride (0.2 mmol) and Et₃N (0.083-0.125 mL, 0.3-0.4 mmol) wasadded, followed by the addition of a solution of 1 mg of active enzyme(0.0037 mmol, 0.037 eq.), as determined by titration withphenylmethanesulfonyl fluoride (PMSF) (Hsia et al., Anal. Biochem.,242:221-227 (1996), which is hereby incorporated by reference), inbuffer solution (10 mmol MES, 1 mmol CaCl₂, pH 5.8). The resulting totalvolume of reaction was 1.0-1.2 mL. The reaction was left stirring atroom temperature for the period of time indicated in Tables 2-4, below.Where D-amino acids were used as acyl donors, after 24 hours, 1 mg moreof active enzyme as well as an equal amount of DMF were added. After thereaction was finished, the mixture was then concentrated in vacuo andsubjected to purification using preparative TLC (5-10% MeOH in CH₂Cl₂).

Peptide Ligation of L-Amino Acids

Acyl donors Z-L-Phe-OBn, Z-L-Ala-OBn, Z-L-Glu-OMe, and Z-L-Lys-SBn (1-4)and acyl acceptors Gly-NH₂ and L-Ala-NH₂ (5, 6) were used for thecoupling reaction as shown in FIG. 5. The acyl donors Z-L-Phe-OBn,Z-L-Ala-OBn, Z-L-Glu-OMe, and Z-L-Lys-SBn (1-4) provided representativeexamples of large and small hydrophobic, negatively charged andpositively charged P₁ side chains, respectively and allowed a broadevaluation of the affinity of the S₁ pocket of these enzymes for variousamino acids. The small amino acid amides Gly-NH₂ and L-Ala-NH₂ (5, 6)were chosen as the acyl acceptors since the S₁′ pocket of subtilisins isnarrow (Moree et al., J. Am. Chem. Soc., 119:3942-3947 (1997); Betzel etal., J. Mol. Biol., 223:427-445 (1992); Sears et al., J. Am. Chem. Soc.,116:6521-6530 (1994); and Jackson et al., Science, 266:243-247 (1994),which are hereby incorporated by reference), and, therefore, it acceptsα-branched amino acids only poorly. The narrow nature of the S₁′ pocketis attributed to the bulky side chain of M222, this residue being aconserved residue amongst subtilisins. Siezen et al., Protein Eng.,4:719-737 (1991), which is hereby incorporated by reference. This haslimited their use in peptide ligation applications.

The coupling of Z-L-Phe-OBn (1), containing the preferred phenylalanineP₁ residue of SBL, with Gly-NH₂ (5) yielded dipeptide Z-L-Phe-Gly-NH₂(7) in excellent yields using both SBL-WT and all four S166-MEs (Table2, below) as catalysts after 1 hour. No reaction was observed in theabsence of the enzyme. M222C—SCH₂CH₂NH3⁺ which had the highestesterase/amidase ratio gave the product in only 33% yield after 5 hoursand starting material, Z-Phe-OBn (1), was recovered in 41% yield. SinceM222C is located at the boundary between S₁ and S₁′ pockets, itsmodification apparently causes steric hindrance at the active siteprecluding substrate binding in the S₁ pocket.

TABLE 2 SBL-WT and SBL-ME-catalyzed coupling of L-amino acids (1-4) andglycinamide (5)^(a) Z-L-Phe-Gly-NH₂ Z-L-Ala-Gly-NH₂ Z-Glu-Gly-NH₂Z-Lys-Gly-NH₂ (7) % yield (8) % yield (9) % yield (10) % yield MEs 1 h 1h 5 h 1 h 5 h 5 h WT 92 68 91 64 62 83 S166C-SCH₂C₆H₅ 92 59 95 68 96 93S166C-SCH₂C₆F₅ 93 42 94 61 61 71 S166C-SCH₂(p-COOH— 100  38 82 30 62 99C₆H₄) S166C-SCH₂CH₂NH₃ ⁺ 95 63 94 69 100  86 M222C-SCH₂CH₂NH₃ ⁺ 33 (5 h)— — — 33 — ^(a)conditions: 0.1 mmol of acyl donor, 0.3 mmol ofglycinamide hydrochloride 0.4 mmol of Et₃N, 1 mg of enzyme, 1:1 H₂O:DMF.The total volume of the reaction is 1.0-1.2 mL.

High yields were also obtained using Z-L-Ala-OBn (2) as the acyl donorwith WT and each of MEs but required a longer reaction time of 5 hours.The yields obtained after running the reaction for 1 hour were all lowerand in all cases starting material was recovered. The requirement for alonger reaction time in this case, compared to using Z-L-Phe-OBn (1) asthe acyl donor, is consistent with SBL-WT's preference for substratebinding of large hydrophobic over small group P₁ substituents in the S₁subsite. Gron et al., Biochemistry, 31:6011-6018 (1992), which is herebyincorporated by reference.

When Z-Glu-OMe (3) with its negatively charged P₁ residue, was used asthe acyl donor, only moderate yields of Z-Glu-Gly-NH₂ (9) were obtainedafter 1 hour in all cases. Unlike the reaction of acyl donor Z-L-Ala-OBn(2), prolonging the reaction time led to an improved yield in the caseof S166C—SCH₂C₆H₅, —SCH₂(p-COOH—C₆H₄), and —SCH₂CH₂NH₃ ⁺. The yieldsusing SBL-WT and S166C—SCH₂C₆F₅ as catalysts after 5 hours werevirtually the same as for 1 hour. However, it was possible to isolateZ-L-Glu-Gly-NH₂ (9) in quantitative yield using S166C—SCH₂CH₂NH₃ ⁺ withits complementary charged S₁ pocket. When using M222C—SCH₂CH₂NH₃ ⁺ asthe catalyst, no enhancement of yield was observed with respect to theSBL-WT catalyzed reaction. This reaction yielded only 33% ofZ-L-Glu-Gly-NH₂ (9) after 5 hours in addition to 32% of the recoveredstarting material Z-L-Glu-OMe (3). As mentioned above, the M222 residueseems to inhibit the binding substrates P₁ residue in S₁ pocket of theenzyme. This may be the cause for the low yield when using this ME andcorrelates with the recovery of starting material. Z-L-Glu-Gly-NH₂ (9)was also obtained in excellent yield (96%) using S166C—SCH₂C₆H₅ as thecatalyst.

Enhanced turnover of the positively charged Z-L-Lys-SBn (4) acyl donorwith the charged ME, S166C—SCH₂(p-COOH—C₆H₄), was observed resulting in99% of Z-L-Lys-Gly-NH₂ (10), the best result of the series. Again, agood yield of 93% of Z-L-Lys-Gly-NH₂ (10) was also observed usingS166C—SCH₂C₆H₅ as the catalyst. This may be due to the highesterase/amidase ratio of this enzyme. The reaction using S166C—SCH₂C₆F₅gave only 71% yield of product which was lower than the reaction usingWT as the catalyst. A higher yield, with respect to WT, was alsoobtained using S166C—SCH₂CH₂NH₃ ⁺ in spite of the potentialelectrostatic repulsion between the modified enzyme and the side chainof Lys.

The synthetic ability of the selected MEs using other acyl acceptorsother than Gly-NH₂ was further investigated. Since the S₁′ pocket ofsubtilisin is small and restricted, the smallest α-branched amino acid,Ala-NH₂ (6), was used to probe this subsite (See Table 3).

TABLE 3 SBL WT and ME-catalyzed coupling of L-amino acids (1-4) andL-alaninamide (6)^(a) Z-L-Lys-Ala- Z-L-Phe-Ala-NH₂ Z-L-Ala-Ala-NH₂Z-L-Glu-Ala-NH₂ NH₂ MEs (11) % yield (12) % yield (13) % yield (14) %yield WT 57 0 0 0 S166C- 51 0 0 0 SCH₂C₆H₅ S166C-SCH₂C₆F₅ 33 0 0 0S166C-SCH₂(p- 48 0 0 0 COOH—C₆H₄) S166C- 88 16 14 0 SCH₂CH₂NH₃ ⁺ M222C-22 0 0 0 SCH₂CH₂NH₃ ⁺ ^(a)conditions: 0.1 mmol of acyl donor, 0.2 mmolof alaninamide hydrochloride, 0.3 mmol of Et₃N, 1 mg of enzyme, 1:1H₂O:DMF, 24 h. The total volume of the reaction is 1.0-1.2 mL.

In all cases, the reaction of L-Ala-NH₂ (6) with Z-L-Phe-OBn (1) wasslower than Gly-NH₂ (5) with Z-L-Phe-OBn (1). Further, after 24 hours,Z-L-Phe-Ala-NH₂ (11) was obtained in moderate yield (33-57%) using WT,S166C—SCH₂C₆H₅, —SCH₂C₆F₅, and —SCH₂(p-COOH—C₆H₄) as the catalysts.However, Z-L-Phe-Ala-NH₂ (11) was obtained in 88% yield in the case ofS166C—SCH₂CH₂NH₃ ⁺. Unlike S166C—SCH₂CH₂NH₃ ⁺, the use ofM222C—SCH₂CH₂NH₃ ⁺ did not improve the yield of the dipeptide product ascompared to the WT-catalyzed reaction; only a low 22% yield ofZ-L-Phe-Ala-NH₂ (11) was obtained. This was possibly due to the stericinteraction of this residue at the binding site, S₁ pocket, with the P₁substrate as mentioned above.

When Z-L-Ala-OBn (2) or Z-L-Glu-OMe (3) were used as acyl donors, noreaction was observed with L-Ala-NH₂ (6) for five out of the six enzymesused. However, when S166C—SCH₂CH₂NH₃ ⁺ was used as the catalyst, theyield of dipeptides Z-Ala-Ala-NH₂ (12) and Z-Glu-Ala-NH₂ (13) wereformed in 16% and 14%, respectively. While these yields were low, theyrepresented a dramatic improvement over WT.

No reaction was observed by treatment of Z-L-Lys-SBn (4) and L-Ala-NH₂(6) with WT and all MEs, including using the negative charged ME,S166C—SCH₂(p-COOH—C₆H₄), in which case the complementary electrostaticinteraction was expected.

These results contrast to the previously reported preference for Alaover Gly in the S₁′ pocket of subtilisin Bacillus lentus. Gron et al.,Biochemistry, 31:6011-6018 (1992), which is hereby incorporated byreference. This preference was not observed: the yields obtained usingglycinamide (5) as the acyl acceptor were higher in all cases, and thereaction times were shorter (Table 2) compared to using alaninamide (6)(Table 3) as the acyl acceptor.

D-Amino Acid Ligation

Next, the scope of application of SBL catalyzed peptide ligation wasextended to include D-amino acid esters Z-D-Phe-OBn, Z-D-Ala-OBn,Z-D-Glu-OMe, Z-D-Lys-OBn, and Ac-D-Phe-OBn (15-19) as the acyl donors(FIG. 6) by the ME methodology, which was not possible with SBL-WT. Theresults are shown in Table 4.

TABLE 4 SBL WT and ME-catalyzed coupling of D-amino acids (15-19) andglycinamide (5)^(a) Z-D-Phe- Z-D-Ala- Z-D-Glu- Z-D-Lys- Ac-D-Phe-Gly-NH₂ Gly-NH₂ Gly-NH₂ Gly-NH₂ Gly-NH₂ MEs (20) % yield (21) % yield(22) % yield (23) % yield (24) % yield WT  0  0 0 0  0 S166C-SCH₂C₆H₅ 6650 3 0 15 S166C-SCH₂C₆F₅ 39 49 3 0 27 S166C-SCH₂(p-COOH— 35 48 6 0 34C₆H₄) S166C-SCH₂CH₂NH₃ ⁺ 43 45 10  0 38 ^(a)conditions: 0.1 mmol of acyldonor, 0.3 mmol of glycinamide hydrochloride, 0.4 mmol of Et₃N, 1 mg ofenzyme, 1:1 H₂O:DMF, 48 h. After 24 h, another 1 mg of enzyme was added.The total volume of the reaction is 1.5-3.0 mL.

For accurate comparison, the D-isomers Z-D-Phe-OBn, Z-D-Ala-OBn,Z-D-Glu-OMe, Z-D-Lys-OBn, and Ac-D-Phe-OBn (15-19) of the representativeL-amino acids Z-L-Phe-OBn, Z-L-Ala-OBn, Z-L-Glu-OMe, and Z-L-Lys-SBn(1-4) examined in the previous ligation examples were used. Thestereoselectivity of SBL-WT for L-amino acids was clear (Table 4),because none of the D-amino acid esters evaluated gave dipeptideproducts with WT as the catalyst. All of the S166C-MEs yielded dipeptideproducts containing D-amino acids Z-D-Phe-Gly-NH₂, Z-D-Ala-Gly-NH₂,Z-D-Glu-Gly-NH₂, Z-D-Lys-Gly-NH₂, and Ac-D-Phe-Gly-NH₂ (20-24). Whileeach of these enzymes still showed a preference for L-amino acids,yields of up to 66% of Z-D-Phe-Gly-NH₂, using S166C—SCH₂C₆H₅, over 0%for WT, demonstrated a dramatic improvement in SBL's acceptance ofD-amino acids.

Similar yields of Z-D-Ala-Gly-NH₂ (21) were obtained using Z-D-Ala-OBn(16) as the acyl acceptor from all four S166C-MEs catalyzed reactions.This demonstrated that chemical modification at this residue broadenedstereospecificity of the S₁ pocket in general manner.

When Z-D-Glu-OMe (17) was used as the acyl donor, only a low yield ofdipeptide Z-D-Glu-Gly-NH₂ (22) was obtained in all ME catalyzedreactions. The best yield, 10% of Z-D-Glu-Gly-NH₂ (22), resulted usingS166C—SCH₂CH₂NH₃ ⁺. This may be accounted for by complementaryelectrostatic interaction between the side chain of S166C—SCH₂CH₂NH₃ ⁺and the side chain of glutamic acid acyl donors. In contrast, no productwas observed when Z-D-Lys-OBn (18) was used as the acyl donor for all WTand ME catalyzed reactions, including the use of S166C—SCH₂(p-COOH—C₆H₄)as the catalyst in which case a great improvement in yield in thereactions of Z-L-Lys-SBn was observed.

Since Z-D-Phe-OBn (15) as the acyl donor gave 35-66% of product witheach of the MEs evaluated and 0% yield with WT and furthermore, sincereplacing the carbobenzoxy (Z) group of Z-D-Phe-OBn (15) with the acetylgroup in the acyl donor Ac-D-Phe-OBn (19) resulted in lower yields(Table 4), it is speculated that the carbobenzoxy group in Z-D-Phe-OBn(15) may direct binding in the S1 pocket as shown in FIG. 7, a processnot observed in Ac-D-Phe-OBn (19).

Dipeptides Produced

Z-Phe-Gly-NH₂ (7) (Moree et al., J. Am. Chem. Soc., 119:3942-3947 (1997)and Morihara et al., Biochem. J., 163:531-542 (1977), which are herebyincorporated by reference): ¹H NMR (CDCl₃) δ3.10 (m, 2H, CH₂Ph), 3.85(2×d, J=2,5 Hz, 2H, NHCH₂CO), 4.40 (m, 1H, NHCHCO), 5.05 (s, 2H,OCH₂Ph), 5.50, 5.70, 6.25, 6.90 (4×brs, 4H, NH), 7.20-7.40 (m, 10H,2×Ph); ¹³C NMR (CDCl₃) δ38.2, 42.7, 56.6, 67.3, 127.2, 128.1, 128.3,128.6, 128.8, 129.2, 135.8, 136.0, 156.3, 171.2, 171.6. HRMS (FAB⁺) MH⁺calcd 356.1610, found 356.1613. [α]³⁰ _(D)=−4.3 (c 0.81, MeOH).

Z-Ala-Gly-NH₂ (8) (Bodanszky et al., Int. J. Peptide Protein Res.,26:550-556 (1985), which is hereby incorporated by reference): ¹H NMR(CDCl₃) δ1.40 (d, J=7 Hz, 3H, CH₃), 3.85 (dd, J=1.7, 5 Hz, 2H, NCH₂CO),4.20 (m, 1H, NHCHCO), 5.10 (dd, J=1.6, 2 Hz, 2H, OCH₂Ph), 5.80, 6.60,7.20 (3×brs, 4H, NH), 7.30-7.40 (m, 5H, Ph); ¹³C NMR (CDCl₃) δ17.7,42.2, 50.6, 66.2, 127.6, 128.0, 136.0,155.9, 171.3, 172.8. HRMS (FAB⁺)MH⁺ calcd 280.1297, found 280.1310. [α]²⁶ _(D)=−8.44 (c 0.97, MeOH);lit. [α]²³ _(D)=−8.5 (c 2, MeOH).

Z-Glu-Gly-NH₂ (9) (Schon et al., Int. J. Peptide Protein Res., 22:92-109(1983), which is hereby incorporated by reference): ¹H NMR (DMSO-d₆)δ1.75-1.95 (m, 2H, CH₂CH₂COOH), 2.30 (m, 2H, CH₂CH₂COOH), 3.65 (dd,J=0.5, 1.7 Hz, 2H, NHCH₂CO), 4.10 (m, 1H, NHCHCO), 5.00 (s, 2H, OCH₂Ph),7.20-7.40 (m, 5H, Ph), 12.40 (brs, 1H, COOH); ¹³C NMR (DMSO-d₆) δ27.2,30.3, 41.9, 54.2, 65.6, 128.4, 128.5, 128.7, 128.8, 136.9, 156.2, 170.8,171.7, 174.0 HRMS (FAB⁺) MH⁺ calcd 338.1352,found 338.1332. [α]²⁸_(D)=−10.2 (c 1.16, MeOH); lit. [α]²⁵ _(D)=−10.2 (c 1.0, MeOH).

Z-L-Lys-Gly-NH₂ (10): ¹H NMR (DMSO-d₆) δ1.50 (m, 2H, CH₂(CH₂)₃NH₂),1.60-1.85 (m, 4H, CH₂CH₂CH₂CH₂NH₂), 2.00-2.18 (m, 2H, (CH₂)₃CH₂NH₂) 3.30(m, 2H, NHCH₂CO), 4.40 (m, 1H, NHCHCO), 5.10 (s, 2H, OCH₂Ph), 6.05, 6.20(2×brs, 2H, NH), 7.20-7.40 (m, 5H, Ph); ¹³C NMR (DMSO-d₆) δ28.1, 29.0,32.2, 42.3, 45.9, 53.8, 66.7, 128.1, 128.6, 136.7, 155.6, 172.2, 175.3.HRMS (FAB⁺) MH⁺ calcd 337.1876, found 337.1842. [α]²⁸ _(D)=+6.97 (c0.55, MeOH).

Z-L-Phe-L-Ala-NH₂ (11) (Morihara et al., Biochem. J., 163:531 -542(1977) and Brubacher et al., Can J. Biochem., 57:1054-1072 (1979), whichare hereby incorporated by reference): ¹H NMR (DMSO-d₆) δ1.20 (d, J=7Hz, 3H, CH₃), 2.90 (m, 2H, CH₂Ph), 4.30 (m, 2H, 2×NHCHCO), 4.90 (s, 2H,OCH₂Ph), 5.75, 6.10, 6.45 (3×brs, 4H, NH), 6.90-7.40 (m, 10H, 2×Ph); ¹³CNMR (DMSO-d₆) δ18.5, 37.4, 48.1, 56.2, 66.2, 126.3, 127.4, 127.7, 128.1,128.3, 129.2, 137.1, 138.2, 155.9, 171.2, 174.1. HRMS (FAB⁺) MH⁺ calcd370.1766, found 370.1753. [α]²⁹ _(D)=−8.86 (c 0.57, MeOH).

Z-L-Ala-L-Ala-NH₂ (12) (Katakai et al., Macromolecules, 6:827-831(1973), which is hereby incorporated by reference): ¹H NMR (DMSO-d₆)δ1.20 (2×d, J=7 Hz, 6H, 2×CH₃), 4.10, 4.20 (m, 2H, 2×NHCHCO), 5.00 (s,2H, OCH₂Ph), 7.20-7.40 (m, 5H, Ph); ¹³C NMR (DMSO-d₆) δ18.1, 18.4, 47.9,50.1, 65.4, 127.7, 128.4, 137.0, 155.8, 172.0. 174.1. HRMS (FAB⁺) MH⁺calcd 294.1454 found 294.1457. [α]²⁵ _(D)=−20.4 (c 0.77, MeOH).

Z-L-Glu-L-Ala-NH₂ (13): ¹H NMR (DMSO-d₆) δ1.20 (d, J=7 Hz, 3H, CH₃),1.82-2.00 (m, 2H, CH₂CH₂COOH), 2.30 (m, 2H, CH₂CH₂COOH), 4.00, 4.20 (m,2H, 2×NHCHCO), 5.00 (s, 2H, OCH₂Ph), 6.20-7.40 (m, 5H, Ph); ¹³C NMR(DMSO-d₆) δ18.4, 26.2, 30.2, 47.9, 53.1, 65.4, 126.5, 127.7, 127.8,128.1, 128.4, 137.0, 156.2, 173.6, 173.8, 174.1. HRMS (FAB⁺) MH⁺ calcd352.1509, found 352.1478. [α]²⁵ _(D)=−16.7 (c 0.76, MeOH).

Z-D-Phe-Gly-NH₂ (20): ¹H and ¹³C NMR data are identical to (7). HRMS(FAB⁺) MH⁺ calcd 356.1610 found 356.1608; [α]³⁰ _(D)=+4.12 (c 1.17,MeOH).

Z-D-Ala-Gly-NH₂ (21) (Richman et al., Int. Peptide Protein Res.,25:648-662 (1985), which is hereby incorporated by reference): ¹H and¹³C NMR data are identical to (8). HRMS (FAB⁺) MH⁺ calcd 280.1297 found280.1298; [α]²⁷ _(D)+10.5 (c 0.72, MeOH); lit. [α]_(D)=+10.5.

Z-D-Glu-Gly-NH₂ (22): ¹H and ¹³C NMR data are identical to (9). HRMS(FAB⁺) MH⁺ calcd 338.1352 found 338.1348; [α]²⁸ _(D)=+10.77 (c 1, MeOH).

Ac-D-Phe-Gly-NH₂ (24) (Thompson et al., J. Med. Chem., 29:104-111(1986), which is hereby incorporated by reference): ¹H NMR (DMSO-d₆)δ1.90 (S, 3H, CH₃), 3.05 (m, 2H, NHCH₂CO), 3.65 (2×d, J=7, 15 Hz, 2H,CHCH₂Ph), 4.40 (q, J=7 Hz, 1H NHCHCO), 7.15-7.25 (m, 5H, Ph); ¹³C NMR(DMSO-d₆) δ22.4, 29.1, 45.9, 54.7, 126.1, 127.9, 128.9, 137.3, 155.2,171.2, 171.5. HRMS (FAB⁺) MH⁺ calcd 264.1348, found 264.1321. [α]³⁰_(D)=−4.38 (c 0.80, MeOH).

Example 5 Peptide Synthesis Using Chemically Modified Mutant Enzymeswith Polar Substituents, Such as Oxazolidinones, Alkyl Amino Groups withPositive Charge, and Saccharides General Methods

¹H and ¹³C NMR spectra were measured on a Varian Unity (400 MHz for ¹Hand 100 MHz for ¹³C) spectrometer with DMSO-d₆ as internal standard.High resolution mass spectra (“HRMS”) were recorded using MicromassZAB-SE (FAB⁺). Optical rotations were measured with a Perkin-Elmer 243Bpolarimeter. ALUGRAM® SIL C/UV254 Art.-Nr. 818 133 (Macherey-Nagel GmbH& Co., Duren, Germany) was used for analytical TLC. Preparative TLC wasperformed on pre-coated Silica gel plate Art.5744 (Merck, Gibbstown,N.J.) visualized with UV light. WT-subtilisin Bacillus lentus and mutantenzymes were purified and prepared as reported in Stabile et al.,Bioorg. Med. Chem. Lett., 6:2501-2506 (1996) and DeSantis et al.,Biochemistry, 37:5968-5973 (1998), which are hereby incorporated byreference, and as described in Example 1. Protected acids were purchasedfrom Sigma-Aldrich Inc. (Milwaukee, Wis.) or Bachem Inc. (Torrance,Calif.) and were used as received. All solvents were reagent grade anddistilled prior to use.

General Procedure for Peptide Ligation

To a solution of Z-L-Phe-OBn (25, 19.2 mg, 0.05 mmol) in DMF (0.25 mL)and water (0.144 mL), glycinamide hydrochloride (31, 17 mg, 0.15 mmol)and Et₃N (0.15 mmol, 0.0625 mL) were added, followed by the addition ofS166C—S-inden-oxaz(S,R) (ME-n, 0.106 mL, 0.5 mg of active enzyme in 10mM MES buffer (pH 5.8) including 1 mM CaCl₂). The reaction was stirredfor one hour at room temperature. The mixture was diluted with AcOEt andwashed with 1 M KHSO₄ (1 mL×1) and brine (1 mL×1), and the organic layerwas dried over MgSO₄. After evaporation, the residue was purified bypreparative TLC (CH₂Cl₂/MeOH=90/10) to afford Z-L-Phe-Gly-NH₂ (33, 17.8mg, quantitative).

Peptide ligations of other substrates using other enzymes were carriedout following the same procedure except for reaction time. In the caseof D-amino acids as acyl donors, 0.5 mg more active enzymes was added tothe reaction vessel after 24 hours, and then the mixture was stirred foranother 24 hours.

Peptide Ligation of L-Amino Acids

First, the coupling reaction of L-amino acid, Z-L-Phe-OBn (25),Z-L-Ala-OBn (26), and Z-L-Glu-OMe (27), with glycinamide (31) wereinvestigated as standard reactions (See FIGS. 1 and 8, Table 5).

TABLE 5 WT and MEs of SBL Catalyzed Peptide Coupling^(a) yield/% acyldonor acyl acceptor product time/h WT ME-j -k -l -m -n -o -p -q -r -s -tZ-L-PheOBn (25) GlyNH₂.HCl (31) Z-L-PheGlyNH₂ (33) 1 92  92 86 88 82100  74 75 95 93 91 95 Z-L-AlaOBn (26) 31 Z-L-AlaGlyNH₂ (34) 5 91  82 8788 91 95 99 91 85 77 92 83 Z-L-GluOMe (27) 31 Z-L-G1uGlyNH₂ (35) 5 62 67 60 54 68 56 63 71 58 65 54 67 Z-D-PheOBn (28) 31 Z-D-PheGlyNH₂ (36)48^(b) 0  9  8 12  7 14  4  4  6  8  7  8 Z-D-AlaOBn (29) 31Z-D-AlaGlyNH₂ (37) 48^(b) 0 61 86 80 86 80 79 73 80 77 72 70 Z-D-GluOBn(30) 31 Z-D-GluGlyNH₂ (38) 48^(b) 0 64 62 60 62 52 74 64 63 62 64 64 25L-AlaNH₂ .HCl (32) Z-L-Phe-L-AlaNH₂ (39) 24^(c) 57  50 31 30 33 37 44 3628 34 31 32 26 32 Z-L-Ala-L-AlaNH₂ (40) 24^(c) 0 10 12 19 21 20 14 11 1516 22 11 27 32 Z-L-Glu-L-AlaNH₂ (41) 24^(c) 0 64 60 59 61 59 58 60 48 5051 55 ^(a)The reaction was performed in DMF/Water (1/1, v/v) using 0.1 Macyl donor, 0.3 M acyl acceptor, and 0.3 M Et₃N in the presence of 0.5mg of active enzyme in 10 mM MES buffer (pH 5.8) containing 1 mM CaCl₂at rt unless otherwise noted. Under same conditions, spontaneoushydrolysis or aminolysis did not occur. ^(b)After 24 h, 0.5 mg of activeenzyme was added and the mixture was stirred for another 24 h. ^(c)Inthese cases, 0.2 M of 32 and 0.2 M of Et₃N were used.

The reactions were carried out using 0.5 mg of active enzyme with Et₃Nin water solution containing 50% DMF. The activities of enzymes weredetermined by titration with phenylmethanesulfonyl fluorine (“PMSF”).Hsia et al., Annal. Biochem., 242:221-227 (1996), which is herebyincorporated by reference. In all cases, the reactions smoothlyproceeded to afford the corresponding dipeptides in good yields. Theseresults indicated that the modification of S166C site by thesesubstituents did not affect the essential ability to accept L-aminoacids in peptide coupling.

Peptide Ligation of D-Amino Acids

Next, the extension of the use of the MEs to the coupling reaction ofD-amino acids as acyl donor, Z-D-Phe-OBn (28), Z-D-Ala-OBn (29), andZ-D-Glu-OBn (30) with Z-L-Phe-OBn (1) was examined. While WT enzyme didnot accept D-amino acids as acyl donors, all of the MEs were able tocatalyze the coupling of D-amino acids with Z-L-Phe-OBn (1). Althoughthe reactions of Z-D-Phe-OBn (28) in all cases were slow to giveZ-D-Phe-Gly-NH₂ (39) in low yield (the best was 14% by using M-n),peptide coupling of Z-D-Ala-OBn (29) or Z-D-Glu-OBn (30) with Gly-NH₂(31) proceeded without remaining substrates. It is noteworthy that usingME-k and -m in case of Z-D-Ala-OBn (29) and ME-o in case of Z-D-Glu-OBn(30) gave Z-D-Ala-Gly-NH₂ (37, 86%) and Z-D-Glu-Gly-NH₂ (38, 74%),respectively, in very high yields. Probably, the CMMs recognized D-aminoacids in a different manner from L-amino acids, i.e., the carbobenzoxygroup of α-position seems to bind the S₁ pocket. On the other hand,repulsion between the phenylmethyl group of Z-D-Phe-OBn (28), which hadthe biggest substituent among the three kinds of substrate, and theother parts in the pocket of active site of the MEs could cause lowreactivity of Z-D-Phe-OBn (28).

In spite of their small S₁′ pocket, all selected MEs were alsoapplicable to the coupling of L-amino acids with an α-branched acylacceptor, L-alaninamide (32). Although the WT enzyme could acceptL-alaninamide (32) as acyl acceptor only in the case of Z-L-Phe-OBn (25)as an acyl donor, the MEs also catalyzed the reactions in the cases ofnot only Z-L-Phe-OBn (25) but also Z-L-Ala-OBn (26) and Z-L-Glu-OMe (27)to afford the corresponding dipeptides Z-L-Phe-L-Ala-NH₂ (39),Z-L-Ala-L-Ala-NH₂ (40), and Z-L-Glu-L-Ala-NH₂ (41), respectively. In thecase of Z-L-Ala-OBn (26), mainly competitive hydrolysis of the esterswas observed (the best was 21% by using ME-m). These results representeda dramatic improvement of the specificity of WT. The yields of thecoupling of Z-L-Glu-OMe (27) with Ala-NH₂ (32) were as good as those ofGly-NH₂ (31) as acyl acceptor, and using ME-j gave the best result(64%). Although not wishing to be bound by theory, it is speculated thatthe strong interaction between the carboxyl group of Z-L-Glu-OMe (27)and the side chain of S166C site of MEs provides a more stableES-complex, which could not be easily attacked by water, thereforeZ-L-Glu-L-Ala-NH₂ (41) could be obtained in good yield.

Dipeptides Produced

Z-L-Phe-Gly-NH₂ (33): ¹H NMR (DMSO-d₆) δ2.74 (dd, J=11.0, 14.0 Hz, 1H,CH₂Ph), 3.04 (dd, J=4.0, 14.0 Hz, 1H, CH₂Ph), 3.59-3.72 (m, 2H,NHCH₂CO), 4.21-4.35 (m, 1H, NHCHCO), 4.93 (d, J=12.5 Hz, 1H, OCH₂Ph),4.94 (d, J=12.5 Hz, 1H), OCH₂Ph), 7.12 (brs, 2H, NH), 7.16-7.38 (m, 5H,Ph), 7.60 (d, J=8.5 Hz, 1H, NH), 8.27 (t, J=5.5 Hz, 1H, NH); ¹³C NMR(DMSO-d₆) δ37.3, 42.0, 56.3, 65.3, 126.3, 127.5, 127.8, 128.1, 128.4,129.3, 137.0, 138.2, 156.0, 170.8, 171.8; HRMS (FAB⁺) calcd forC₁₉H₂₂N₃O₄ (M+H)⁺356.1610, found 356.1639; [α]²¹ _(D)=−3.94 (c 1.04,MeOH).

Z-L-Ala-Gly-NH₂ (34): ¹H NMR (DMSO-d₆) δ1.20 (d, J=7.0 Hz, 3H, CH₃),3.60 (dd, J=5.5, 16.0 Hz, 1H, CH₂NH), 3.62 (dd, J=5.5, 16.0 Hz, 1H,CH₂NH), 4.03 (dq, J=7.0, 7.0 Hz, 1H, CH₃CHNH), 5.00 (d, J=12.5 Hz, 1H,OCH₂Ph), 5.03 (d, J=12.5 Hz, 1H, OCH₂Ph), 7.11 (brs, 1H, NH₂), 7.18(brs., 1H, NH₂), 7.27-7.42 (m, 5H, Ph), 7.57 (d,J=7.0 Hz, 1H, NH), 8.11(t, J=5.5 Hz, 1H, NH); ¹³C NMR (DMSO-d₆) δ17.9, 42.0, 50.3, 65.5,127.85, 127.89, 128.4, 136.9, 155.9, 170.9, 172.7; HRMS (FAB⁺) calcd forC₁₃H₁₈N₃O₄ (M+H)⁺280.1297, found 280.1307; [α]²⁵ _(D)=−8.44 (c 0.64,MeOH).

Z-L-Glu-Gly-NH₂ (35): ¹H NMR (DMSO-d₆) δ1.66-1.79 (m, 1H, CH₂CH₂COOH),1.83-1.95 (m, 1H, CH₂CH₂COOH), 2.26 (t, J=7.5 Hz, 2H, CH₂COOH), 3.62 (d,J=5.5 Hz, 2H, NHCH₂CO), 3.95-4.05 (m, 1H, NHCHCO), 5.01 (d, J=12.5 Hz,1H, OCH₂Ph), 5.03 (d, J=12.5 Hz, 1H, OCH₂Ph), 7.07 (brs, 1H, NH), 7.20(brs, 1H, NH), 7.25-7.40 (m, 5H, Ph), 7.55 (d, J=7.5 Hz, 1H, NH), 8.11(t, J=5.5 Hz, 1H, NH); 12.20 (brs, 1H, COOH); ¹³C NMR (DMSO d₆) δ27.0,30.2, 41.9, 54.1, 65.6, 127.8, 127.9, 128.4, 136.9, 156.2, 170.8, 171.7,174.0; HRMS (FAB⁺) calcd for C₁₅H₂₀ ⁻N₃O₆ (M+H)⁺338.1352, found338.1364; [α]²⁵ _(D)=−9.28 (c 0.69, MeOH).

Z-D-Phe-Gly-NH₂ (36): HRMS calcd for C₁₉H₂₂N₃O₄ (M+H)⁺356.1610, found356.1592; [α]²¹ _(D)=+3.42 (c 1.17, MeOH).

Z-D-Ala-Gly-NH₂ (37): HRMS calcd for C₁₃H₁₈N₃O₄ (M+H)⁺280.1297, found280.1303; [α]²⁴ _(D)=+8.49 (c 0.86, MeOH)

Z-D-Glu-Gly-NH₂ (38): HRMS calcd for C₁₅H₂₀N₃O₆ (M+H)⁺338.1352, found338.1353 ; [α]²⁴ _(D)=+9.07 (c 1.08, MeOH)

Z-L-Phe-L-Ala-NH₂ (39): ¹H NMR (DMSO-d₆) δ1.22 (d, J=7.0 Hz, 3H, CH₃),2.71 (dd, J=13.5, 13.5 Hz, 1H, CH₂Ph), 3.03 (dd, J=3.5, 13.5 Hz, 1H,CH₂Ph), 4.18-4.31 (m, 2H, NHCHCO×2). 4.93 (s, 2H, OCH₂Ph), 7.04 (brs,1H, NH), 7.14-7.22 (m, 1H, NH), 7.55 (d, J=8.5 Hz, 1H, NH), 8.08 (d,J=7.5 Hz, 1H, NH); ¹³C NMR (DMSO-d₆) δ18.5, 37.4, 48.1, 56.2, 65.2,126.3, 127.4, 127.7, 128.1, 128.4, 129.3, 137.1, 138.2, 155.9, 171.1,174.1; HRMS (FAB⁺) calcd for C₂₀H₂₄N₃O₄ (M+H)⁺370.1767, found 370.1769;[α]²⁴ _(D)=−8.86 (c 0.44, MeOH).

Z-L-Ala-L-Ala-NH₂ (40): ¹H NMR (DMSO-d₆) δ1.19 (d, J=7.0 Hz, 3H, CH₃),1.24 (d, J=7.5 Hz, 3H, CH₃), 3.90-4.26 (m, 2H, NHCHCO×2), 5.01 (s, 2H,CH₂OPh), 7.02 (brs, 1H, NH), 7.13 (brs, 1H, NH), 7.25-7.45 (m, 5H, Ph),7.51 (d, J=6.5 Hz, 1H, NH), 7.88 (d, J=7.5 Hz, 1H, NH); ¹³C NMR(DMSO-d₆) δ18.1, 18.5, 47.9, 50.2, 65.4, 127.78, 127.84, 128.4, 137.1,155.8, 172.0, 174.2; HRMS (FAB⁺) calcd for C₁₄H₂₀N₃O₄ (M+H)⁺294.1454,found 294.1457; [α]²¹ _(D)=−20.4 (c 0.77, MeOH).

Z-L-Glu-L-Ala-NH₂ (41): ¹H NMR (DMSO-d₆) δ1.20 (d, J=8.0 Hz, 3H, CH₃),1.68-1.82 (m, 1H, CH₂CH₂COOH), 1.82-2.03 (m, 1H, CH₂CH₂COOH), 2.21-2.40(m, 2H, CH₂CH₂COOH), 3.93-4.25 (m, 2H, NHCHCO×2), 5.02 (s, 2H, OCH₂Ph),7.02 (brs, 1H, NH), 7.18-7.46 (m, 5H, Ph), 7.54 (dd, J=7.5, 24.0 Hz, 2H,NH₂), 7.92 (d, J=7.5 Hz, 1H, NH), 12.40 (brs, 1H, COOH); ¹³C NMR(DMSO-d₆) δ18.4, 26.2, 30.3, 48.0, 53.1, 65.4, 127.0, 127.7, 127.8,127.9, 128.4, 129.3, 137.0, 156.2, 173.7, 173.8, 174.1; HRMS (FAB⁺)calcd for C₁₆H₂₂N₃O₆ (M+H)⁺352.1509, found 352.1502; [α]²⁵ _(D)=−16.7 (c0.76, MeOH).

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 2 <210> SEQ ID NO 1 <211> LENGTH: 269<212> TYPE: PRT <213> ORGANISM: Bacillus lentus <220> FEATURE:<223> OTHER INFORMATION: Subtilisin SBL <400> SEQUENCE: 1Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Va #l Gln Ala Pro Ala Ala 1               5   #                10   #                15His Asn Arg Gly Leu Thr Gly Ser Gly Val Ly #s Val Ala Val Leu Asp            20       #            25       #            30Thr Gly Ile Ser Thr His Pro Asp Leu Asn Il #e Arg Gly Gly Ala Ser        35           #        40           #        45Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gl #y Asn Gly His Gly Thr    50               #    55               #    60His Val Ala Gly Thr Ile Ala Ala Leu Asn As #n Ser Ile Gly Val Leu65                   #70                   #75                   #80Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Va #l Lys Val Leu Gly Ala                85   #                90   #                95Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gl #n Gly Leu Glu Trp Ala            100       #           105       #           110Gly Asn Asn Gly Met His Val Ala Asn Leu Se #r Leu Gly Ser Pro Ser        115           #       120           #       125Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Se #r Ala Thr Ser Arg Gly    130               #   135               #   140Val Leu Val Val Ala Ala Ser Gly Asn Ser Gl #y Ala Gly Ser Ile Ser145                 1 #50                 1 #55                 1 #60Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Va #l Gly Ala Thr Asp Gln                165   #               170   #               175Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gl #y Ala Gly Leu Asp Ile            180       #           185       #           190Val Ala Pro Gly Val Asn Val Gln Ser Thr Ty #r Pro Gly Ser Thr Tyr        195           #       200           #       205Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pr #o His Val Ala Gly Ala    210               #   215               #   220Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Tr #p Ser Asn Val Gln Ile225                 2 #30                 2 #35                 2 #40Arg Asn His Leu Lys Asn Thr Ala Thr Ser Le #u Gly Ser Thr Asn Leu                245   #               250   #               255Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Al #a Thr Arg            260       #           265 <210> SEQ ID NO 2<211> LENGTH: 275 <212> TYPE: PRT<213> ORGANISM: Bacillus amyloliquefaciens <220> FEATURE:<223> OTHER INFORMATION: Subtilisin BPN′ <400> SEQUENCE: 2Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Il #e Lys Ala Pro Ala Leu 1               5   #                10   #                15His Ser Gln Gly Tyr Thr Gly Ser Asn Val Ly #s Val Ala Val Ile Asp            20       #            25       #            30Ser Gly Ile Asp Ser Ser His Pro Asp Leu Ly #s Val Ala Gly Gly Ala        35           #        40           #        45Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gl #n Asp Asn Asn Ser His    50               #    55               #    60Gly Thr His Val Ala Gly Thr Val Ala Ala Le #u Asn Asn Ser Ile Gly65                   #70                   #75                   #80Val Leu Gly Val Ala Pro Ser Ala Ser Leu Ty #r Ala Val Lys Val Leu                85   #                90   #                95Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Il #e Ile Asn Gly Ile Glu            100       #           105       #           110Trp Ala Ile Ala Asn Asn Met Asp Val Ile As #n Met Ser Leu Gly Gly        115           #       120           #       125Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Va #l Asp Lys Ala Val Ala    130               #   135               #   140Ser Gly Val Val Val Val Ala Ala Ala Gly As #n Glu Gly Thr Ser Gly145                 1 #50                 1 #55                 1 #60Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Ty #r Pro Ser Val Ile Ala                165   #               170   #               175Val Gly Ala Val Asp Ser Ser Asn Gln Arg Al #a Ser Phe Ser Ser Val            180       #           185       #           190Gly Pro Glu Leu Asp Val Met Ala Pro Gly Va #l Ser Ile Gln Ser Thr        195           #       200           #       205Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gl #y Thr Ser Met Ala Ser    210               #   215               #   220Pro His Val Ala Gly Ala Ala Ala Leu Ile Le #u Ser Lys His Pro Asn225                 2 #30                 2 #35                 2 #40Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Gl #u Asn Thr Thr Thr Lys                245   #               250   #               255Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Le #u Ile Asn Val Gln Ala            260       #           265       #           270 Ala Ala Gln        275

What is claimed:
 1. A method of producing a modified subtilisin enzymehaving esterase and amidase activities, said method comprising providinga modified enzyme wherein a cysteine residue is substituted for an aminoacid residue selected from the group consisting of residues 62, 166, 217and 222, wherein said amino acid residue is numbered according to itsequivalent in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ (SEQ ID NO: 2), and wherein said cysteine residue ismodified by replacing the thiol hydrogen in the cysteine residue with athiol side chain selected from the group consisting of ——SCH₃,——SCH₂CH_(3,) ——SCH₂CH(CH₃)_(2,) ——S(CH₂)₄CH_(3,) —S(CH₂)₅CH_(3,)——S(CH₂)₉ CH₃——SCH₂ C₆H_(5,) ——SCH_(2,) CH_(2,) NH₃+, ——SCH_(2,) CH_(2,)SO₃−, ——SCH_(2,) (p-COOH——C₆H_(4,)), and ——SCH _(2,)C₆ F₅ to form themodified enzyme.
 2. A method according to claim 1, wherein the esteraseactivity is from about 350 s⁻¹ mM⁻¹ to about 11100 s⁻¹ mM⁻¹.
 3. A methodaccording to claim 1, wherein the amidase activity is from about 5.6 s⁻¹nM⁻¹ to about 355 s⁻¹ mM⁻¹.
 4. A method according to claim 1, whereinthe enzyme is a protease.
 5. A method according to claim 4, wherein theprotease is a Bacillus lentus subtilisin.
 6. A method according to claim1, wherein the amino acid replaced with a cysteine is an amino acidselected from the group consisting of asparagine, leucine, methionine,and serine.
 7. A method according to claim 1, wherein the amino acidreplaced with a cysteine is in a subsite of the enzyme.
 8. A methodaccording to claim 7, wherein the subsite is selected from the groupconsisting of S₁, S₁′, and S₂.
 9. A method according to claim 1, whereinthe thiol side chain is selected from the group consisting of—SCH₂(p-COOH—C₆H₄) and —SCH₂C₆F₅.
 10. A method according to claim 9,wherein the thiol side chain is —SCH₂(p-COOH—C₆H₄).
 11. A methodaccording to claim 9, wherein the thiol side chain is —SCH₂C₆F₅.